Solubilizing steroidal drugs by β-cyclodextrin derivatives

Abstract

Administration of steroidal drugs is hampered by their very low solubilities in water. β-Cyclodextrin and β-cyclodextrin derivatives can solubilize steroids and improve bio-availability of these hydrophobic APIs. A systematic overview of the achievable solubility enhancements of various steroids, testosterone, estradiol, progesterone, hydrocortisone, prednisone, dexamethasone, and finasteride, is provided. Beside the spatial fit of the steroid within the cyclodextrin cavity also hydrophilic substituents at the cyclodextrin framework play an important role in the extent of solubilization observed. Uniformly substituted anionic heptakis-6-sulfoethylsulfanyl-6-deoxy-β-cyclodextrin (HSES) performed best, reaching complexation efficiencies of 60–90 mol% for most steroids. Two neutral β-cyclodextrin thioethers, heptakis-6-methylsulfanyl-6-deoxy-2-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)]-β-CD (HTMT) and heptakis-6-thioglyceryl-6-deoxy-β-CD (HTG) showed gender selectivity in binding of hormons: HTMT was selective for testosterone, while HTG was selective for estradiol. Solubilization is mainly due to complexation of the A and B rings as well as C and D rings of the steroid framework as demonstrated by ROESY NMR spectroscopy.

Introduction

Steroids are polycyclic isoprenoids derived from cholesterol consisting of four fused rings from which three are six-membered (A, B and C) and one is five-membered (D). In all living organisms, specific functions are controlled by steroids. The sex steroids, also called gonadal steroids, are responsible for the development and control of sexual characteristics. They include the female sex hormones estradiol and progesterone and the male sex hormone testosterone (Ettinger et al., 2016, Katznelson et al., 1996). Furthermore, glucocorticoids such as cortisone control inflammation and immune response (Steinhilber, 2005), while mineralocorticoids like aldosterone influence body fluid and electrolyte balance (Sharp and Leaf, 1966).

Steroids are generally lipophilic being nearly insoluble in water. Because of their lipophilicity steroids can solely pass the blood brain barrier (Pardridge and Mietus, 1979). On the other hand, they require transport aids liquids, such as serum albumin or specific hormone binding globulins, to move in the aqueous body (Anderson, 1974; Daughaday, 1957; Slaunwhite and Sandberg, 1958; Södergård et al., 1982).

Beside their natural occurrence steroids became increasingly interesting as active pharmaceutical ingredients (APIs) within the last years. Among the gonadal steroids, testosterone is administered in the so-called testosterone replacement therapy to elderly men who suffer disorders like erectile dysfunction, metabolic syndrome and reduction in muscle mass (Salehian et al., 1995b, Zitzmann et al., 2006). The female sex hormones estradiol and progesterone are also applied to compensate hormone deficiencies of women during menopause leading to the climacteric syndrome (Cauley et al., 1995, Cohen et al., 2003). Administration of gonadal steroids remains problematic because uptake via the gastro-intestinal tract is hampered by their very low solubility in water (Brewster et al., 1988). Testosterone is, therefore, better transdermally administered from hydrogels containing ethanol as a solubilizer of the steroid (An et al., 2003). Delivery of progesterone is even more challenging since it is even much less water-soluble. Because of the very low solubilities, it remains difficult to control the administered dose of the gonadal steroid precisely. This uncertainty gives rise to some chance of overdose. Since overdose of sex steroids can lead to severe side effects like prostate, breast or liver cancer (Ahmed et al., 1988, Vom Saal et al., 1997), present hormone replacement therapy is accompanied by some risks which should be diminished by a better solubilization of these APIs.

Delivery of glucocorticoids like hydrocortisone is less critical than the one of the gonadal steroids since glucocorticoids are better soluble in water due to additional hydroxy groups at the steroid framework. Glucocorticoids are important APIs to constrain inflammation, allergy and immune response. Transplantation of organs would not be possible without the application of glucocorticoids (Steinhilber, 2005). Glucocorticoids are transported within the human body by glucocorticoid binding globulin as well as by serum albumin (Daughaday, 1957, Slaunwhite and Sandberg, 1958). Hydrocortisone is administered as a microcrystal suspension through inhalation or intravenous injection (Langlands and McNeill, 1960, Scarfone et al., 1995). Microemulsions of glucocorticoids are applied for transdermal delivery as well (Lehmann et al., 2001).

Beside the natural steroid hormones also many synthetic APIs derived from the steroid framework are in use. Dexamethasone is indeed a more potent synthetic analog of hydrocortisone, but it is less soluble in water (Meikle and Tyler, 1977). Another example is finasteride which is applied for the treatment of benign prostatic hyperplasia and pattern hair loss of males (Duijnhoven et al., 2014, Irwig and Kolukula, 2011). The action of finasteride is based on the inhibition of type II and type III 5α-reductase which converts testosterone into dihydrotestosterone (Yamana et al., 2010). Since an overdose of finasteride can cause side effects like erectile dysfunction and depression, the dosage of finasteride must be controlled properly (Irwig and Kolukula, 2011). Despite of its low solubility in water finasteride is administered orally but transdermally as well (Gormley, 1995, Tabbakhian et al., 2006).

In general, exact dosage of steroidal APIs appears to be problematic because of their hydrophobicity. Transdermal delivery is presently still the most efficient and reliable way of delivery. Mostly hydrogels are in use for this purpose which requires an organic co-solvent like ethanol or DMSO to solubilize the steroid. Since daily exposition of the skin to ethanol over an extended period of time might be harmful, alternative excipients would be desirable. Microemulsions are indeed an alternative, but the necessary content of detergents might also cause irritations of the skin (Kogan and Garti, 2006). Furthermore, cyclodextrins are also able to solubilize hydrophobic APIs in water avoiding the side effects mentioned above.

Cyclodextrins (CDs) are cyclic α(1→4)-linked oligomers of glucose which are produced by enzymatic degradation of maize starch in industrial scale (Wimmer, 2000). Three ring sizes are commercially available in pharmaceutical purity: CDs consisting of 6, 7, or 8 glucose units, called α-, β-, and γ-CD (Wenz, 1994). While α-CD and γ-CD are regarded as non-toxic, and they are allowed as novel food ingredient by the European Commission (Dalli, 2012, Munro et al., 2004, Vassliou, 2008), β-CD is known to be harmful to erythrocytes at high concentrations because of extraction of cholesterol from the cell wall (Kiss et al., 2010). Furthermore β-CD is less soluble in water (1.85 wt.% at room temperature) than α-CD (14.5 wt.%) and γ-CD (23.2 wt.%) (Szejtli, 1998). As shown in Table 1 derivatization is advantageous for the improvement of aqueous solubility and reduction of toxicity of β-CD (Kiss et al., 2010). Most commonly used derivatives of β-CD are randomly substituted hydroxypropyl-β-CD (HP-β-CD, Cavasol W7 HP, Wacker Chemie AG, Germany) and sulfobutyl-β-CD (SBE-β-CD, Captisol, Ligand, USA), which are more than 50 wt.% water-soluble and less harmful (Rajewski et al., 1995, Szente and Szejtli, 1999). Despite these obvious advantages, these CD derivatives are mixtures of various degrees of substitution (d.s.) and substitution patterns. These uncertainties impede batch-to-batch reproducibility and quality control. Highly water-soluble methylated derivatives of β-CD are also known, statistically substituted ones with around 12–13 methyl groups (RAMEB) as well as uniform ones, heptakis-(2,6-di-O-methyl)-β-CD (DIMEB) with 14 methyl groups and heptakis-(2,3,6-tri-O-methyl)-β-CD (TRIMEB) with 21 methyl groups. Cholesterol extraction capability increases with increasing number of methyl groups reaching a maximum for 14 methyl groups. As a consequence, hemolytic activity of DIMEB is highest with a very low concentration for 50% hemolytic activity (→Table 1) HC₅₀ = 0.9 mM, equivalent to 0.12 wt.% (Kiss et al., 2010).

Another group of uniformly substituted derivatives is thio ethers solely attached at all primary positions of CDs. They are readily obtainable from per-6-bromo-6-deoxy- or per-6-iodo-6-deoxy-CDs through nucleophilic displacement reactions by thiols (Becker et al., 2014, Mazzaglia et al., 2001, Steffen et al., 2007). Octakis-6-carboyethylsulfanyl-6-deox-γ-CD (Sugammadex, Bridion, Merck Sharp & Dohme, USA) is the most prominent example since it is commercialized as an agent for reversal of neuromuscular blocking caused by the steroidal anesthetic rocuronium (Bom et al., 2002, de Boer et al., 2006). Furthermore, neutral anionic and cationic thioethers of β-CD are known, which show high solubilities in water (Becker et al., 2014, Mazzaglia et al., 2001, Steffen et al., 2007. Three of them, heptakis-6-sulfoethylsulfanyl-6-deoxy-β-CD (HSES), heptakis-6-methylsulfanyl-6-deoxy-2-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)]-β-CD (HTMT), and heptakis-6-thioglyceryl-6-deoxy-β-CD (HTG) had been also included in Table 1. Three commercial available CDs, β-CD, HP-β-CD, and SBE7-β-CD and three thioether substituted derivatives, HSES, HTMT, and HTG had been selected for our investigations (→ Scheme 1).

CDs are called host molecules since they are able to complex guest molecules in aqueous media (Wenz, 1994). Main driving forces for this kind of supramolecular self-organization are the hydrophobic effect, the expulsion of high energy water from the hydrophobic CD cavity, and dipole-dipole interactions (Biedermann et al., 2014, Schneider, 2017). Most often complexes with 1:1 stoichiometry are formed, but also sometimes complexes with 1:2 or 2:1 stoichiometry (host/guest). The equilibrium constants, so-called binding constant, K of most 1:1 complexes range between 10 and 10,000 M⁻¹ (Houk et al., 2003, Rekharsky and Inoue, 1998). Binding depends on the spatial fit of the guest within the CD cavity and on the pattern of substituents of the CD derivative. The more completely the hydrophobic guest is filling the cavity, the higher will be the binding constant (Fourmentin et al., 2013). Substituents at the secondary side generally reduce the binding potential of the host, because they prevent the formation of intramolecular hydrogen bonds which otherwise rigidify the cavity (Wenz, 2012). In contrast, substitution of the primary hydroxy groups by the more hydrophobic thioether groups improves the binding potential so that exceptional high binding constants (K > 10⁶ M⁻¹) can be reached (Thiele et al., 2011, Wenz et al., 2008).

Because most of the hydrophobic part of a guest can be masked through complexation, hydrophobic guests can be solubilized in water by CDs. If a 1:1 complex is formed, the solubility of the guest increases linearly with the concentration of the host till the solubility of the host-guest complex is reached. The slope of this so-called solubility isotherm s corresponds to the molar ratio of complexed guest per total host (Connors, 1997, Connors and Pendergast, 1984).

Meanwhile there are many drug formulations on the market using β-CD hosts to improve bioavailability of the API including piroxicam/β-CD (Brexin, Ciesi, Italy), omeprazole/β-CD (Omebeta, Betapharm, Germany), Itraconazole/HP-β-CD (Sporanox, Janssen, Germany), Voriconazole/SBE-β-CD (Vfend, Pfizer, Germany) (Loftsson and Duchene, 2007, Wenz, 2000). The internal diameter of β-CD of 0.58 to 0.65 nm is suitable to accommodate steroids (Müller and Wenz, 2007). Complexation of cholesterol leads to the hemolytic activity of β‐CD and its methylated derivatives as mentioned above. Dissolution of cholesterol plaques, so-called cholesterol depletion, by means of methylated β-CD was already discussed for the treatment of Alzheimer’s disease (Simons et al., 1998).

The gonadal steroid testosterone was complexed in β-CD (Salehian et al., 1995a). The complex of testosterone in HP-β-CD is better soluble in water. It was already marketed in the USA (Andotest-SL, Savient Pharmaceuticals, USA) for sublingual application (Wang et al., 1996). β-Estradiol complexed in DIMEB (Aerodiol, Servier, France) was sold for nasal application (Schipper et al., 1990). Also, glucocorticoids could be solubilized by β‐CD derivatives. Complexation of hydrocortisone by HP-β-CD allows transdermal delivery without using alcohol. In classic formulations of hydrocortisone isopropyl myristate and isopropyl alcohol show a penetration-enhancing effect. (Brinkmann and Müller-Goymann, 2003). Permeation rates could be significantly enhanced by this host (Chang and Banga, 1998). Also, hydrocortisone complexed in HP-β-CD was marketed (Dexocort, Actavis, Iceland) and the synthetic steroid dexamethasone was complexed in β-CD (Glymesason, Fujinaga, Japan) and HP-β-CD for eye drops against conjunctivitis (Loftsson and Stefánsson, 2002).

Binding constants of different steroids to β-CD derivatives are already published (Albers and Müller, 1992; Asbahr et al., 2009; Frasconi and Mazzei, 2009; Lahiani-Skiba et al., 2006; Liu et al., 1990; Usayapant et al., 1991), but these data are difficult to compare because solubility and binding constant strongly depend on the ionic strength of the solvent, due to the salting out effect (Loftsson et al., 2005). In addition, the measured binding constant also depends partially on the method of determination (Rekharsky and Inoue, 1998). The highest binding constant was observed for the most hydrophobic steroid progesterone in β-CD, 24,705 M⁻¹ (Liu et al., 1990), the lowest for finasteride in HP-β-CD, 673 M⁻¹ (Asbahr et al., 2009).

The work consisted of the determination of phase-solubility profiles of seven steroidal drugs in the presence of various β-CD derivatives at low concentrations in an aqueous buffered medium. To simulate physiological condition, an isotonic HEPES buffer at pH 7.4 was chosen.

Four natural steroids testosterone, β‐estradiol, progesterone and hydrocortisone and three synthetic steroids prednisone, dexamethasone and finasteride had been selected for our investigations (→Scheme 2).